Symbiotic fixation of atmospheric nitrogen

ABSTRACT

inoculated cells from plants are caused to grow aseptically in a synthetic liquid culture medium to produce a mass of undifferentiated plant cells. These are aseptically incoulated with a symbiotic microorganism to produce a synthetic symbiotic system that is capable of fixing atmospheric nitrogen. The end product contains available protein material.

United States Patent Hardy et al.

[451 Dec. 5, 1972 Richard D. Holsten, Wilmington, Del.

[73] Assignee: E. I. du Pont de Nemours 8: Company, Wilmington, Del.

[22] Filed: June 4, 1971 [21] Appl. No.: 150,179

Related U.S. Application Data [63] Continuation-impart of Ser. No.805,382, March 7,

1969, abandoned.

[52] U.S. Cl ..47/58 [51] Int. Cl. ..A01h 17/00 [58] Field of Search...l95/50; 47/58, 1.2; 71/1, 6-10 [56] References Cited UNlTED STATESPATENTS 2,747,334 5/1956 Routien et al. ..47/58 3 ,514,900 6/1970 McDade..47/58 3,628,287 12/1971 Staba et al ..47/58 1 OTHER PUBLICATIONS Lewiset al., Botanical Gazette 95: pages 316-329 relied on.

Primary Examiner-Robert E. Bagwill Attorney-Anthony P. Mentis [57]ABSTRACT inoculated cells from plants are caused to grow aseptically ina synthetic liquid culture medium to produce a mass of undifferentiatedplant cells. These are aseptically incoulated with a symbioticmicroorganism to produce a synthetic symbiotic system that is capable offixing atmospheric nitrogen. The end product contains available proteinmaterial.

3 Claims, No Drawings SYMBIOTIC FIXATION OF ATMOSPHERIC NITROGEN CROSSREFERENCE TORELATED APPLICATION Arachis hypogea Rhizobium .rp. Pisum.rativum Rhizobium leguminosarum Trifalium pratensi Rhizobium lrifoliiNutrient materials which favor the growth of plant This application is acontinuation-impart of our 5 cells are well known. They encompass theso-called copending application Ser. No. 805,382, filed Mar. 7, 1969,and now abandoned.

The fixation of atmospheric nitrogen is highly important both as anatural and a commercial process. Symbiotic nitrogen fixation as itoccurs in nature is a primary factor in agriculture and food productionand any method which can augment this natural process is potentiallyvaluable. Considerable effort has been expended in the search forsynthetic methods of fixing nitrogen but no completely satisfactoryprocess has yet been found. A system whereby relatively large quantitiesof undifferentiated plant cells can be grown in a restricted space andused to convert atmospheric nitrogen into utilizable reduced nitrogencompounds such as proteins is an important goal.

It is an object of this invention to obtain fixation of nitrogen by asynthetic process involving the aseptic production and growth ofundifferentiated plant cells together with symbiotic microorganisms.

It is a further object of this invention to obtain symbioticnitrogen-fixation which involves incubating undifferentiated plant cellsunder aseptic conditions with symbiotic microorganisms in aliquid-culture medium so that the microorganisms penetrate and occupythe plant cells.

lt is a still further object of this invention to obtain development ofthe undifferentiated plant .cells with their contained symbioticmicroorganisms in a medium which contains few if any exogenous growthfactors.

These and other objects of the invention are accomplished by providing aliquid nutrient medium containing live undifferentiated plant cells,aseptically inoculating the plant cells in the liquid culture mediumwith live symbiotic microorganisms, maintaining the liquid culturemedium in aseptic condition to allow the microorganisms to enter intoand occupy the plant cells, and for the undifferentiated plant cellswith their contained micro-organisms to fix gaseous nitrogen from theatmosphere.

A mass of undifferentiated plant cells may be obtained by asepticallyremoving a small piece of plant tissue from a selected organ, as fromthe root, stem, etc. and placing it in a sterile medium containingappropriate nutrients. Such a tissue explant will grow and proliferateinto a large number of the same type of plant cells or of related plantcells, withoutspecialization of these cells to form specific plantorgans such as roots or leaves, etc. These cells may be referred to as aheterogeneous population or colony of undifferentiated plant cellscomprised of single cells as well as aggregates of cells. This type ofuninterruptedcell growth and multiplication without the formation ofspecific plant organs is known as undifferentiated cell growth.

Many symbiotic combinations capable of fixing atmospheric nitrogen areknown to exist in nature, including the Leguminosae family of plants andtheir symbiotic soil bacteria. Some examples are:

Medicaga saliva Rhizobium meliloti plant growth factors, inorganicsalts, including salts of trace elements, carbohydrates, and vitamins.All of these may be of natural or synthetic origin. The plant growthfactors include cytokinins such as deproteinized coconut milk,benzyladenine and the like; and auxins, such as2,4-dichlorophenoxyacetic acid, indoleacetic acid, indolebutyric acid,naphthaleneacetic acid and so forth. The inorganic salts, which areherein referred to as basal salts, include phosphates, nitrates,chlorides and sulfates, etc. of metals such as sodium, potassium,calcium, magnesium and of metals which are needed in trace amounts only.Carbohydrates include glucose, sucrose, starch, corn syrup and the likewhile vitamins may be illustrated by thiamine, riboflavin, pantothenicacid, niacin and so forth. Carbohydrates and the mineral salts areusually considered to be nutrients for bacteria. The amounts of thesenutrient materials which may be used are well known and may be variedquite widely so long as sufficient quantities are used to favor thegrowth of the organisms involved.

The medium in which the inoculation of the undifferentiated plant cellswith the microorganisms takes place must be liquid, e.g., water, inwhich the nutrient materials are contained. This is a most importantelement since it appears to be necessary for the bacteria to invade theplant cells. In the absence of a liquid medium the bacteria do not enterinto the plant cells and no symbiotic relation is established.

In general the present invention contemplates a completely asepticprocedure in which the first step is the aseptic growth ofundifferentiated plant cells derived from members of the Leguminosae ina suitable medium. With the undifferentiated plant cells contained in aliquid medium having the necessary nutrient materials present, bacteriaof the genus Rhizobium are aseptically added. The plant cells andbacteria in the liquidculture medium are then incubated together for asuitable time and at a suitable temperature, usually about 20 to about30C., to allow microorganisms to enter and occupy the plant cells.Further growth and development of the plant cells with their containedmicroorganisms is accomplished in .a medium having substantially nogrowth stimulating activity on the plant cells. The greatestnitrogen-fixing activity occurs in this latter stage.

The final products containing plant cells and symbiotic bacteria arevaluable sources of useable reduced nitrogen formed from the normallyunuseable nitrogen of the atmosphere. These finalproducts are valuableas foods or food supplements containing proteins or amino acids forlivestock such as swine and poultry or other animals which must besupplied with reduced nitrogen and/or amino acids. The products can alsoserve as a source from which valuable nitrogen compounds such asproteins and essential amino acids can be extracted according to knownmethods. The reduced nitrogen products can further be used asnitrogenfertilizers either alone or admixed with other known fertilizercompositions.

I MMO 3 an extent that eventually the microbesessentially fill theentire plant cell. Electron microscope photographs show the plant cellcytoplasm is almost entirely replaced by the microorganism population.Invasion of undifferentiated plant cells by microorganisms under thesynthetic conditions of this invention has never been reportedpreviously and appears to be a new phenomenon.

A culture of the appropriate plant tissue can be established byaseptically removing a small piece of the organ of selected cell originand placing this tissue explant upon the surface of an agar-solidifiedbasal medium containing plant growth factors. The natural growthfactors, e.g., deproteinized coconut milk, can be supplied in theconcentration range of 2 to 20 percent by volume; cytokinins in themolar concentration range of to 10'; auxins in .the molar concentrationrange of 10' to 10', with various combinations of these factors beingutilized to initiate rapid cell divisions in the selected plant tissue.Following establishment of a mass of rapidly growing, undifferentiatedcells, which may be referred to as a callus, on the agar medium, thecells are transferred to a liquid system employing a growth mediumsimilar in composition to that utilized to initiate the primary culture.Continued rapid cell divisions in this liquid system give rise to alarge undifferentiated plant cell population which is then inoculatedwith the appropriate symbionic microorganisms.

The plant cells and microorganisms are kept in intimate association inthe liquid system in the dark for a period of about 2 to 10 days whichis usually sufficient to allow initial penetration of the plant cells bythe bacteria.

After bacterial penetration and invasion of the plant cells has beenaccomplished, the cells are washed with sterile basal salts solution orsterile water, etc. to remove any extra-cellular microorganisms ormetabolic products which might interfere with the continued developmentof the symbiotic relationship. The washed cells are then transferred tofresh sterile growth media composed of: (a) basal salts, (b) basal saltsand cytokinins, or (c) basal salts, cytokinins, and auxins. Thesemediamay be either liquid or solid (e.g., with agar). The cultures aregenerally maintained for several days in a temperature range of -30C.for further development of the symbiosis.

The symbiotic system thus established fixes atmospheric nitrogen asdetermined by using the reduction of acetylene to ethylene assay,byextraction of cellular proteins and by fixation of "'N isotope, appliedto selected undifferentiated cell samples. The nitrogenfixing activityof the cultures is related to the plant growth factors in the culturemedia as well as to the degree of microorganism invasion of the plantcells. Reduction of exogenous cytokinins and auxins stimulates bothmicroorganism penetration of the cells and nitrogen-fixing activity.

The following examples illustrate how the invention may be practiced.

EXAMPLE I Cultures of soybean root tissue (Glycine max'var. Acme) wereinitiated by germinating surface-sterilized seed under asepticconditions on an agar-solidified basal medium containing the following:

BASAL MEDIUM Ingredients mgJliter Nl-LNO 1650.0 KNO, 1900.0 KH,PO l70.0Na,EDTA (sodium ethylene diamine tetraacetic acid) 37.3 FeS0 '7H,O 27.8"3 6.2 MI'ISO4'4HQO ZnSO,-4H,O 8.6

0.83 Na,Mo0 -2H,O 0.25 CuSOrSl-LO 0.025 CoCl '6H,O 0.025 CaCl,-2H,O440.0 MgS0 -7H,O 370.0 Thiamine HCl 0.4 lnositol 100.0 Sucrose 30,0000

After the seeds had germinated in about 1 week and when each primaryroot had reached a length of approximately 2 inches, it was excisedusing aseptic techniques and a small (approximately 5 mm) segment orexplant was removed from the middle of each root and placed on thesurface of an agar-solidifed basal medium in a closed container. Thismedium was as described above except that it was supplemented with 15percent deproteinizedcoconut milk (CM) and 2 mg. per liter of2,4-dichloro-phenoxyacetic acid (2,4-D). Theroot explants wereincubatedat about 25C. in the dark to initiate cell proliferation and toproduce a mass of growing undifferentiated plant cells or callus. Thecallus mass, composed of undifferentiated, actively dividing cells, wasthen aseptically subcultured to fresh liquid media of similarcomposition to maintain growth of the cells. The liquid cultures wereaerated by continuous shaking, tumbling, or other appropriate means asby bubbling a stream of sterile air through the liquid. Following theestablishment of an actively growing undifferentiated cell culture,liquid transfers were made to fresh media of varying compositions atselected intervals. A mass of undifferentiated cells derived fromsoybean root growing in 225 ml. of liquid medium in the dark, whichcontained the components shown in the preceding table together with 15percent deproteinized coconut milk and 2 ppm (parts per million)2,4-dichlorophenoxyacetic acid, was inoculated with 0.5 ml of a liquidculture of soil bacteria identified as Rhizobium japonicum (ATCC 10324or Strain No. 61A76; The Nitragin Co.). The bacterial culture medium hadthe following composition (in grams/liter):

The bacteria were maintained in the flask with the plant cells for aperiod of 5 days to permit invasion of the plant cells by the bacteria.After 5 days, the mass of undifferentiated plant cells and containedbacteria was removed from the culture flask, aseptically washed with abasal salts medium and transferred to fresh liquid and agar-solidifiedmedia of the following compositions:

I. Basal medium 2. Basal medium 10% coconut milk 3. Basal medium coconutmilk 2 ppm 2,4-D

The agar-solidified cultures, one from each treatment, were grown in thelight, about 1,000 foot candles from fluorescent tubes, as well as inthe dark. The liquid cultures, 50 ml. contained in 250 ml. Erlenmeyerflasks, were incubated for 16 days at room temperature under laboratorylight conditions with constant shaking to facilitate aeration. The lightgrown cultures on semisolid agar media were also maintained at roomtemperature.

After 12 days of incubation on the semi-solid agar medium in the light,the cultures were assayed for acetylene to ethylene conversion activityas proof of their nitrogen-fixing ability. The assay was performed byplacing the plant cells from the culture into a 5 ml. glass syringe. Thesyringe was stoppered with a rubber stopall, flushed with anargonzoxygen (08:02 by volume) atmosphere and finally gassed with 5 ml.of an Ar:O :C b42 atmosphere of composition 0.7:0.2:0.1 by

Millimicromoles of ethylene I Undifferentiated produced per Plant CellsCulture Treatment sample in 24 hours. fresh wt. (mg.)

Basal medium 5.6 76.9 Basal medium 10% coconut milk 4.3 117.6 Basalmedium 10% coconut milk 2 ppm 2.4-D 0.7 90.7

The ethylene production was highest in the basal medium alone and felloff with increasing growth factor additions. The most active mass ofplant cells was the least in fresh weight.

Samples of plant cells from the preceding were fixed in aformalinzacetic acidzethyl alcohol solution (5:5:90 by volume),dehydrated through the ethanolseries and finally embedded in paraffinfor sectioning and study of tissue morphology. The analysis of theseplant cell masses shows there is greatest bacterial invasion of theplant cells in the basal medium along. Lesser bacterial invasion occurswhere the basal medium is supplemented with growth factors and both ofthese findings correlate with the acetylene to ethylene activity.

EXAMPLE II Millimicromoles of ethylene Undifferentiated produced perPlant Cells Culture Treatment sample in 24 hours. fresh wt. (mg) Basalmedium 14,4 45.2

Basal medium 10% coconut milk 5.6 43.4 Basal medium 10% coconut milk 2ppm 2,4-D 3.6 95.1

The highest acetylene to ethylene activity was found in theunsupplemented cultures and decreasing activity in the cultures havingadded growth factors. Paraffin sections showed greater bacterialinvasion in those cells kept in the unsupplemented basal medium, withlesser amounts of bacterial invasion in those cells kept in the basalmedium to which growth factors had been added.

EXAMPLE III The inoculated plant cells of Example I which weretransferred to liquid media and allowed to incubate at room temperaturewith constant shaking for 16 days, were also analyzed for acetylene toethylene activity. The following results were obtained withsubstantially equivalent weights of tissue:

Millimicromoles of ethylene produced per Culture Treatment sample in.24hours. Basal medium 3.8 Basal medium 10% coconut milk 1.2 Basal medium10% coconut milk 2 ppm 2,4-D 0.4

The acetylene to ethylene activity was in direct proportion to thereduction in growth factor additions and was lower overall.

EXAMPLE lV Undifferentiated plant cells which are not inoculated withbacteria show negligible acetylene to ethylene activity in this assaysystem. In an experiment, undifferentiated plant cells of soybean rootorigin, both bacterially infected and non-infected, growing in a liquidbasal medium with cytokinin and auxin supplements were analyzed foracetylene to ethylene activity in the standard assay proceduce using 50ml. syringes. The following results were obtainedat 24 hours ofincubation at room temperature:

Millimicromoles of ethylene produced per g. dry weight Culture Treatmentsample in 24 hours Undifferentiated plant cells Rhiz. japonicum 87.5Undifferentiated plant cells (no bacteria) 16.5

EXAMPLE V The total protein content of soybean root tissue grown inliquid culture as described in Example 1 either inoculated withsymbiotic bacteria or uninoculated was determined using the Biuretprocedure. The culture inoculated with Rhizobium japonicum strain 61A76was maintained on a medium containing only minimal quantities ofinorganic nitrogen compounds as shown in the tabulation below while theuninoculated control culture received the full nitrogen basal mediumdescribed in Example 1, supplemented with 15 percent whole coconut milkand 2 mgJliter of 2,4-D.

The tissue samples for protein determination were ground in a mortar andpestle using 10 ml. of distilled water per sample. The homogenate wasfiltered to remove cellular debris and 2 ml. of the filtrate was treatedwith 0.5 ml. of 50 percent trichloroacetic acid to precipitate theproteins. The proteinaceous precipitate was collected as a pellet bycentrifuging for 1 hour at 3,000 rpm in a clinical centrifuge. The clearsupernatant above the pellet was removed by pipette and discarded.

The resultant pellets were treated with 2 ml. of biuret reagent and 2ml. of distilled water. The tubes were incubated at 37C. in a water bathfor 30 minutes before determining the optical density at'540 mp. againsta reagent blank. The total protein content was determined by comparingthe optical density (O.D. with a standard curve. The following resultswere obtained for total protein in each sample:

Fresh Protein in sample wt. (g) mg. Inoculated tissue 2.09 15 .05 0.72Uninoculated tissue 2.81 5.90 0.20

The'assay shows that utilizable amounts of protein are extractable fromtissue-cultures grown in liquid culture, and that inoculation of thetissues with a symbiotic bacterium leads to markedly increased levels ofprotein over those in the uninoculated tissue even though the lattersystem was supplied with higher levels of reduced nitrogen in theculture medium.

Low Nitrogen Medium (pl-l 5.8)

Ingredients EXAMPLE VI Undifferentiated cells from soybean root tissue(Acme variety) as'described in Example I were grown in air in a lownitrogen liquid medium (see Example V) supplemented with 10' p.benzyladenine and 2 mg./liter indoleacetic acid. The cultures weremaintained in the dark for about 2 weeks to obtain substantial growth.The cultures were then inoculated with Rhizobium japonicum strain 61A76for establishment of the symbiotic relationship, with a control culturemaintained on a similar medium but not inoculated with bacteria.

After 7 days of incubation following the day of bacterial inoculation,cellular invasion by the Rhizobia was apparent. At this time the airatmosphere of the 80 ml. flasks was replaced with 0.15 atm oxygen, 0.60atm argon and 0.25 atm N-enriched nitrogen gas (99.9 atom N). Incubationwas continued for 10 days after sealing the flasks. All flasks weretreated in the manner described.

On the 10th day the tissue was removed, weighed and subjected toKjeldahl digestion with'the resulting ammonia isolated as (NI-[Q 80 bydistillation by standard procedures. Using the procedures described byBurris and Wilson (Methods in Enzymology, Vol. IV, 1957) the isolated(NH SO was subjected to treatment with alkaline bromine and the Ngenerated by this treatment collected and analyzed for masses 28, 29 and30 by mass spectrometric analysis. From these data the extent of Nincorporation was calculated from atom N enrichment values. The resultsobtained (0.016 atom N) for thesymbiotic cultures as shown below exceedthe minimum value'considered by Burris and Wilson as confirming fixationof this isotope.

The above test is based on the fact that in nature nitrogen comprises99.636% N isotope and 0.364% "N isotope and this ratio will be found inall nitrogen containing materials, including air, unless the materialhas been enriched with oneor the other isotope. A sample of air isanalyzed to provide the numerical air standard value from which possibleenrichments can be determined by difference and also to insure that thespectrometer is functioning properly.

In the procedures illustrated the cellular growth media, andparticularly the basal media, are of a standard and well recognized.type that supply nutrients necessary for cells. One such medium usedfor cultivation of plant tissue is shown in US. Pat. No. 2,747,334.

The concentration of the various components can vary within relativelywide limits.

The temperatures employed are those for isolated cellular growth,usually from 20 to 30C. and preferably about 2327C.

The process of this invention can be carried out either in the presenceor absence of light and it is generally preferred to conduct the growthof the undifferentiated plant cells in the presence of air.

Although undifferentiated plant cells derived from soybean root tissueand Rhizobium japonicum are shown in the examples, other plant andbacterial species which are capable of producing a symbioticnitrogen-fixing system are applicable, as previously stated. As oneexample, when peanut cotyledon tissue is used with Strain No. 47Al (TheNitragin Co.) Rhizobium, grown in substantially the same media as theRhizobium of Example 1, assays show that nitrogenfixing activity isestablished.

The media employed should be produced and maintained under substantiallyaseptic conditions, i.e., substantially free from other living organismsexcept those rapid cell division with some dependence on the particularplant species studied. The coconut milk is a source of cytokinins andauxins which are required for normal plant cell growth. In tissueculture methodology, the molar concentration ranges utilized for thecytokinins vary from approximately 10' to 10", and for the auxins, 10 to10''. The examples show 2,4- dichlorophenoxyacetic acid as an auxin at 2mg./liter for optimum growth.

It has been reported that the ratio of moles of ethylene formed in theacetylene assay to moles of nitrogen fixed is between 3 and 4. Usingthis range, the following calculations of the millimicromoles ofnitrogen fixed per sample for each example were made.

TABLE example 1 Millimicromoles of nitrogen Culture Treatment fixed persample in 24 hrs.

Basal medium 1.40 to 1.86 Basal medium 10% coconut milk 1.07 to 1.43Basal medium 10% coconut milk 2 ppm 2,4-D 0.17 to 0.23

Example [I Basal medium 3.60 to 4.80 Basal medium 10% coconut milk 1.40to 1.87 Basal medium 10% coconut milk 2 ppm 2,4-D 0.90 to 1.20

Example 111 Millimicromoles of nitrogen Culture Treatment fixed persample in 24 hrs.

Bacterial penetration into the plant cells takes place in from 2 to 10days in the liquid environment, with 3 to 5 days generally beingsatisfactory. One to two weeks of additional time are usually sufficientto produce the greatest nitrogen-fixing activity. The actual number ofbacterial cells added can vary widely. In the preceding example, about300 X cells were added per 225 ml. of culture medium. This number doesnot appear to be critical and the only requirement seems to be for anactively growing bacterial culture specific for the strain of plantcells used.

It has been previously shown that nitrogen-fixing activity is directlyrelated to acetylene reducing activity of symbionts. The quantitativerelationship between dicated by the eqitiagionsz nitrogen-fixing andacetylene-reducing activity is in- N KZNH CgHgfiCgH;

Ethylene produced by this reduction is readily determined by gaschromatographic analysis.

Since obvious modifications and equivalents in the inventionwill beevident to those skilled in the involved arts, we propose to be boundsolely by the appended claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:

1. A process for synthetic, aseptic, symbiotic fixation of gaseousnitrogen consisting essentially in the steps of a. providing a liquidnutrient culture medium consisting essentially of live undifferentiatedplant cells from the family Leguminosae;

b. aseptically inoculating the undifferentiated plant cells in theliquid culture medium with live symbiotic microorganisms from the genusRhizobium;

c. maintaining the liquid culture medium in aseptic condition in thepresence of gaseous nitrogen to allow the microorganisms to enter andoccupy the undifierentiated plant cells;

d. aseptically washing the plant cells to remove extracellularmicroorganisms and metabolic products; and

. maintaining the washed plant cells and their contained microorganismsin a sterile liquid nutrient medium, which has substantially no growthstimulating activity on the said plant cells, to allow the plant cellswith their contained symbiotic microorganisms to fix gaseous nitrogen.

2. A process according to claim 1 in which the liquid culture medium ofstep (a) contains added plant growth factors.

3. A process for fixing gaseous nitrogen consisting essentially in thesteps of providing an aqueous culture medium consisting essentially ofundifferentiated plant cells derived from the family Leguminosae,

said culture medium containing a plurality of nutrient materialsselected from the group consisting of plant growth factors, mineralsalts, carbohydrates and vitamins, in quantities sufficient to favorgrowth of the plant cells,

aseptically inoculating the undifferentiated plant cells in the aqueousculture medium with live soil bacteria of the genus Rhizobium,

maintaining the inoculated culture medium in aseptic condition for about2 to 10 days at a temperature of about 20C. to 30C. to allow thebacteria to enter into the plant cells,

aseptically transferring the undifferentiated plant cells and theircontained bacteria to a sterile nutrient culture medium substantiallyfree of exogenous plant growth factors, and

aseptically maintaining the culture medium for a further period of aboutone to two weeks in the presence of gaseous nitrogen to allow the systemto fix gaseous nitrogen.

"\umn Al in

2. A process according to claim 1 in which the liquid culture medium ofstep (a) contains added plant growth factors.
 3. A process for fixinggaseous nitrogen consisting essentially in the steps of providing anaqueous culture medium consisting essentially of undifferentiated plantcells derived from the family Leguminosae, said culture mediumcontaining a plurality of nutrient materials selected from the groupconsisting of plant growth factors, mineral salts, carbohYdrates andvitamins, in quantities sufficient to favor growth of the plant cells,aseptically inoculating the undifferentiated plant cells in the aqueousculture medium with live soil bacteria of the genus Rhizobium,maintaining the inoculated culture medium in aseptic condition for about2 to 10 days at a temperature of about 20*C. to 30*C. to allow thebacteria to enter into the plant cells, aseptically transferring theundifferentiated plant cells and their contained bacteria to a sterilenutrient culture medium substantially free of exogenous plant growthfactors, and aseptically maintaining the culture medium for a furtherperiod of about one to two weeks in the presence of gaseous nitrogen toallow the system to fix gaseous nitrogen.